Na/K-ATPase in intercalated cells along the rat nephron revealed by antigen retrieval

The Na/K-ATPase plays a fundamental role in the physiology of various mamma lian cells. In the kidney, previous immunocytochemical studies have localiz ed this protein to the basolateral membrane in different tubule segments. H owever, intercalated cells (IC) of the collecting duct (CD) in rat and mous e were unlabeled with anti-Na/K-ATPase antibodies. An antigen retrieval tec hnique has been recently described in which tissue sections are pretreated with sodium dodecyl sulfate before immunostaining. This procedure was used to reexamine the presence of Na/K-ATPase in IC along the rat nephron using monoclonal antibodies against the Na/K-ATPase alpha-subunit. Subtypes of IC along the nephron were identified by their distinctive staining with polyc lonal and monoclonal antibodies to the 31-kD vacuolar H+-ATPase subunit, wh ereas principal cells (PC) were labeled with a polyclonal antibody to the w ater channel aquaporin-4 (AQP-4). In PC, the Na/K-ATPase and AQP-4 staining colocalized basolaterally. In contrast to previous reports, we found that IC of all types showed basolateral labeling with the anti-Na/K-ATPase antib ody. The staining was quantified by fluorescence image analysis. It was wea k to moderate in IC of cortical and outer medullary collecting ducts and mo st intense in IC of the initial inner medullary collecting duct. IC in the initial inner medulla showed a staining intensity that was equivalent or st ronger to that in adjacent principal cells. Models of ion transport at the cellular and epithelial level in rat kidney, therefore, must take into acco unt the potential role of a basolateral Na/K-ATPase in intercalated cell fu nction.